Antigen-mediated exocytosis in RBL-2H3 cells is associated with substantial hydrolysis of membrane inositol phospholipids and an elevation in concentration of cytosol Ca2+ ((Ca2+)). Studies with intact cells suggested that elevation of cytosol Ca2+ and activation of protein kinase C provide necessary signals for secretion. A casual relationship between these two events and secretion has now been established in studies with permeabilized RBL-2H3 cells. It is shown that cells appropriately labeled with (3H)myoinositol or (3H)-5HT release (3H)inositol phosphates or (3H)-5HT when stimulated with antigen or GTPgS at buffered concentrations of free Ca(2+) (0.1-1 muM). The phorbol esterase, PMA, which activates protein kinase C, causes release of 5-HT but does not increase the hydrolysis of phosphatidyl inositol. Furthermore release of both (3H)inositol phosphates and (3H)-5HT can be inhibited in parallel by neomycin which sequesters inositol phospholipids and by GDPbetaS which competes with GTP for binding to G-proteins. Another consequence of activation of protein kinase C is the activation of the Na+/H+ antiport system which in turn elevates the pH of the cytosol in RBL-2H3 cells. The specific roles of Ca2+, pH and protein kinase C in the mediation of secretion was studied with permeabilized cells in which it was shown that the requirement for Ca2+ with either for antigen - or GTPgS-induced secretion can be reduced from 1 uM to O.1 uM if the pH of the buffer was increased from 7.0 to 7.4. These data suggest that in intact cells secretion can be maintained at low (Ca2+) once the intracellular pH of the cells has increased.